Pharmaceutical composition for inhibiting the growth of viruses and cancers

ABSTRACT

This invention is a method of treating cancer, both carcinomas and sarcomas, and viral infections, in particular HIV through the administration of a pharmaceutical composition containing a benzimidazole derivative. The composition is also claimed. The benzimidazole derivative is selected from the group consisting of:  
                 
 
     wherein X is hydrogen, halogen, alkyl of less than 7 carbon atoms or alkoxy of less than 7 carbon atoms; n is a positive integer of less than 4; Y is hydrogen, chlorine, nitro, methyl, ethyl or oxychloro; R is hydrogen, alkylaminocarbonyl wherein the alkyl group has from 3 to 6 carbon atoms, or an alkyl group of from 1 to 8 carbon atoms and R 2  is 4-thiazolyl, NHCOOR 1  wherein R 1  is aliphatic hydrocarbon of less than 7 carbon atoms, prodrugs, pharmaceutically acceptable salts and mixtures thereof and a pharmaceutically acceptable carrier

[0001] This is a continuation of application Ser. No. 10/106,429, filedMar. 26, 2002, which is a continuation of U.S. Ser. No. 09/748,651,filed Dec. 22, 2000, now U.S. Pat. No. 6,362,207 issued Mar. 26, 2002,which is a divisional application of U.S. Ser. No. 09/264,942, filedMar. 9, 1999, now U.S. Pat. No. 6,262,093, which is acontinuation-in-part application of U.S. Ser. No. 08/927,550, filed Sep.6, 1997, now U.S. Pat. No. 5,880,144, which is a divisional of U.S. Ser.No. 08/771,193, filed Dec. 20, 1996, now U.S. Pat. No. 5,767,138, andwhich is a divisional of U.S. Ser. No. 08/420,914, filed Apr. 12, 1995,now abandoned. Application U.S. Ser. No. 09/264,942 is also acontinuation-in-part of U.S. Ser. No. 09/081,384, filed May 19, 1998,now abandoned, and also a continuation-in-part of U.S. Ser. No.09/081,627, filed May 19, 1998, now abandoned.

TECHNICAL FIELD

[0002] This invention is a method of treating cancer, both carcinomasand sarcomas, and viral infections, in particular HIV through theadministration of a pharmaceutical composition containing abenzimidazole derivative. The composition is also claimed.

BACKGROUND OF THE INVENTION

[0003] Cancers are the leading cause of death in animals and humans. Theexact cause of cancer is not known, but links between certain activitiessuch as smoking or exposure to carcinogens and the incidence of certaintypes of cancers and tumors has been shown by a number of researchers.

[0004] Many types of chemotherapeutic agents have been shown to beeffective against cancers and tumor cells, but not all types of cancersand tumors respond to these agents. Unfortunately, many of these agentsalso destroy normal cells. The exact mechanism for the action of thesechemotherapeutic agents are not always known.

[0005] Despite advances in the field of cancer treatment the leadingtherapies to date are surgery, radiation and chemotherapy.Chemotherapeutic approaches are said to fight cancers that aremetastasized or ones that are particularly aggressive. Such cytocidal orcytostatic agents work best on cancers with large growth factors, i.e.,ones whose cells are rapidly dividing. To date, hormones, in particularestrogen, progesterone and testosterone, and some antibiotics producedby a variety of microbes, alkylating agents, and anti-metabolites formthe bulk of therapies available to oncologists. Ideally cytotoxic agentsthat have specificity for cancer and tumor cells while not affectingnormal cells would be extremely desirable. Unfortunately, none have beenfound and instead agents which target especially rapidly dividing cells(both tumor and normal) have been used.

[0006] The development of materials that would target tumor cells due tosome unique specificity for them would be a breakthrough. Alternatively,materials that were cytotoxic to tumor cells while exerting mild effectson normal cells would be desirable.

[0007] HIV and other viral infections are another leading cause ofdeath. HIV is a disease in which a virus is replicated in the body whichattacks the body's immune system. The HIV virus is not easily destroyednor is there a good mechanism for keeping the host cells fromreplicating the virus. Herpes Simplex is another viral infection whichis difficult, if not impossible, to cure. A method of treating thesediseases and other viral infections is highly desirable. A materialwhich would target the HIV virus and inhibit viral replication is highlydesirable.

[0008] The benzimidazole derivatives used herein to treat cancer and/orviral infection have been used as fungicides and as antihelmetics.

SUMMARY OF THE INVENTION

[0009] A method of treating cancer, in particular, treating cancers inwarm blooded animals and humans, comprising administering atherapeutically effective amount of a composition comprising abenzimidazole compound selected from the group consisting of:

[0010] wherein X is hydrogen, halogen, alkyl of less than 7 carbon atomsor alkoxy of less than 7 carbon atoms; n is a positive integer of lessthan 4; Y is hydrogen, chlorine, nitro, methyl, ethyl or oxychloro; R ishydrogen, alkylaminocarbonyl wherein the alkyl group has from 3 to 6carbon atoms, or an alkyl group of from 1 to 8 carbon atoms and R₂ is4-thiazolyl, NHCOOR₁ wherein R₁ is aliphatic hydrocarbon of less than 7carbon atoms, and preferably an alkyl group of less than 7 carbon atomsis claimed. Preferably the compositions contain:

[0011] wherein R is an alkyl of 1 through 8 carbon atoms and R₂ isselected from the group consisting of 4-thiazolyl, NHCOOR₁, wherein R₁is methyl, ethyl or isopropyl and the non-toxic, pharmaceuticallyacceptable acid salts with both organic and inorganic acids. The mostpreferred compounds are 2-(4-thiazolyl)benzimidazole,methyl-(butylcarbamoyl)-2-benzimidazolecarbamate and2-methoxycarbonylamino-benzimidazole and those wherein Y is chloro.

[0012] In the present invention it has been discovered that thecompounds described above are useful for the inhibition of HIV and thetreatment of HIV infection and similar retrovirus infections. Thepresent invention also provides methods for the treatment of HIVinfection comprising administering to a host infected with HIV apharmaceutically or therapeutically effective or acceptable amount of acompound as described above, particularly those wherein R is4-thiazolyl.

[0013] The present invention also provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound as described above.

[0014] These compositions have been discovered to inhibit the growth ofcancer or other tumors in humans or animals and to induce apoptosis ofcancer cells by administration of a therapeutically effective amount ofthe composition, preferably by administering a benzimidazole compound tothe site of the cancer.

[0015] More specifically, this invention provides an anti-cancercomposition comprising a pharmaceutical carrier and a benzimidazolederivative as defined herein along with a method for treating suchcancers. These compositions can induce apoptosis in cancer cells.

[0016] These compositions are also effective against viruses and areused to treat viral infections and this invention provides a method oftreating viral infections such as herpes, hepatitis, influenza andrhinoviruses.

DETAILED DESCRIPTION OF THE INVENTION

[0017] A. Definitions:

[0018] As used herein, a “pharmaceutically acceptable” component is onethat is suitable for use with humans and/or animals without undueadverse side effects (such as toxicity, irritation, and allergicresponse) commensurate with a reasonable benefit/risk ratio.

[0019] As used herein, the term “safe and effective amount” refers tothe quantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.By “therapeutically effective amount” is meant an amount of a compoundof the present invention effective to yield the desired therapeuticresponse. For example to inhibit HIV infection or treat the symptoms ofinfection in a host or an amount effective to delay the growth of or tocause a cancer, either a sarcoma or lymphoma, to shrink. The specificsafe and effective amount or therapeutically effective amount will, varywith such factors as the particular condition being treated, thephysical condition of the patient, the type of mammal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulations employed and the structure of thecompounds or its derivatives.

[0020] As used herein, a “pharmaceutical salts” is salt of thebenzimidazole derivatives which are modified by making acid or basesalts of the compounds. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids. Preferably the salts are made using an organic orinorganic acid. These preferred acid salts are chlorides, bromides,sulfates, nitrates, phosphates, sulfonates, formates, tartrates,maleates, malates, citrates, benzoates, salicylates, ascorbates, and thelike.

[0021] As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering thebenzimidazole derivatives to the animal or human. The carrier may beliquid or solid and is selected with the planned manner ofadministration in mind.

[0022] As used herein, “cancer” refers to all types of cancer orneoplasm or malignant tumors found in mammals, including carcinomas andsarcomas. Examples of cancer are cancer of the brain, breast, cervix,colon, head & neck, kidney, lung, non-small cell lung, melanoma,mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.

[0023] As used herein, the “benzimidazole derivatives” are thebenzimidazoles, and their salts and also their prodrugs. The exactbenzimidazoles are described in detail below. The preferred materialsare the products sold under the names “Thiabendazole®”, “Benomyl®” and“Carbendazim®” by BASF and Hoechst, DuPont and MSD-AgVet.

[0024] As used herein “viruses” includes viruses which infect animals ormammals, including humans. Viruses includes retorviruses, HIV,influenza, polio viruses, herpes, herpes simplex, rhinoviruses,hepatitis, and the like.

[0025] B. The Benzimidazole Derivatives

[0026] The benzimidazole derivatives which are known for theirantifungal activities. They are systemic fungicides used to prevent anderadicate fungi. The compounds have the following structure:

[0027] wherein X is hydrogen, halogen, alkyl of less than 7 carbon atomsor alkoxy of less than 7 carbon atoms; n is a positive integer of lessthan 4; Y is hydrogen, chlorine, nitro, methyl, ethyl or oxychloro; R ishydrogen, alkylaminocarbonyl wherein the alkyl group has from 3 to 6carbon atoms or an alkyl group having from 1 to 8 carbons, and R₂ is4-thiazolyl, NHCOOR₁ wherein R₁ is aliphatic hydrocarbon of less than 7carbon atoms, and preferably and alkyl group of less than 7 carbonatoms. Preferably the compositions are:

[0028] wherein R is an alkyl of 1 through 8 carbon atoms and R₂ isselected from the group consisting of 4-thiazolyl, NHCOOR₁, wherein R₁is methyl, ethyl or isopropyl and the non-toxic, pharmaceuticallyacceptable acid salts with both organic and inorganic acids.

[0029] The most preferred compounds are 2-(4-thiazolyl)benzimidazole,methyl-(butylcarbamoyl)-2-benzimidazolecarbamate and2-methoxycarbonylamino-benzimidazole and the compounds wherein Y ischloro and X is hydrogen.

[0030] The benzimidazole compounds also include prodrugs. “Prodrugs” areconsidered to be any covalently bonded carriers which release the activeparent drug according to the formula of the benzimidazole derivativesdescribed above in vivo when such prodrug is administered to a mammaliansubject. Prodrugs of the benzimidazole compounds are prepared bymodifying functional groups present in the compounds in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compounds. Prodrugs include compounds whereinhydroxy, amine, or sulfhydryl groups are bonded to any group that, whenadministered to a mammalian subject, cleaves to form a free hydroxyl,amino, or sulfhydryl group, respectively. Examples of prodrugs include,but are not limited to, acetate, formate, or benzoate derivatives ofalcohol and amine functional groups in the benzimidazole derivatives;phosphate esters, dimethylglycine esters, aminoalkylbenzyl esters,aminoalkyl esters and carboxyalkyl esters of alcohol and phenolfunctional groups in the benzimidazole derivatives; and the like.

[0031] The pharmaceutically acceptable salts of the benzimidazolederivatives include the conventional non-toxic salts or the quaternaryammonium salts of the benzimidazole derivatives formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include those derived from inorganic acidssuch as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric and the like; and the salts prepared from organic acids such asacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric,citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

[0032] The pharmaceutically acceptable salts of the present inventionare synthesized from the benzimidazole derivatives which contain a basicor acidic moiety by conventional chemical methods. Generally, such saltsare prepared by reacting the free acid or base forms of these compoundswith a stoichiometric amount of the appropriate base or acid in water orin an organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference. The disclosures of all of the references cited herein arehereby incorporated herein by reference in their entirety.

[0033] Synthesis

[0034] The benzimidazole derivatives are prepared in a number of wayswell known to one skilled in the art of organic synthesis. Thebenzimidazole derivatives are synthesized using the methods describedbelow, together with synthetic methods known in the art of syntheticorganic chemistry, or variations thereon as appreciated by those skilledin the art. Preferred methods include but are not limited to thosemethods described below. Each of the references cited below are herebyincorporated herein by reference.

[0035] These compounds are prepared according to the method described inU.S. Pat. No. 3,738,995 issued to Adams et al, Jun. 12, 1973. Thethiazolyl derivatives are prepared according to the method described inBrown et al., J. Am. Chem. Soc., 83, 1764 (1961) and Grenda et al., J.Org. Chem., 30, 259 (1965).

[0036] C. Dosage and Dosage Delivery Forms

[0037] The type of compound and the carrier and the amount will varywidely depending on the species of the warm blooded animal or human,body weight, and tumor being treated. The dosage administered will varydepending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the age, health and weight of the recipient; the natureand extent of the symptoms; the kind of concurrent treatment; thefrequency of treatment; and the effect desired.

[0038] The benzimidazole is preferably micronized or powdered so that itis more easily dispersed and solubilized by the body. Processes forgrinding or pulverizing drugs are well known in the art. For example, ahammer mill or similar milling device are used. The preferred particlesize is less than about 100μ and preferably less than 50μ.

[0039] The dosage administered will vary depending upon known factorssuch as the pharmacodynamic characteristics of the particular activeingredient, and its mode and route of administration; age, sex, health,metabolic rate, absorptive efficiency and/or weight of the recipient;nature and extent of symptoms; kind of concurrent treatment, frequencyof treatment; and the effect desired.

[0040] A “tumor growth inhibiting amount” of the benzimidazolederivatives is that amount which is effective to inhibit or slow thegrowth of a tumor.

[0041] Dosage forms (compositions) suitable for internal administrationcontain from about 1.0 milligram to about 5000 milligrams of activeingredient per unit. In these pharmaceutical compositions, the activeingredient will ordinarily be present in an amount of about 0.5-95% byweight based on the total weight of the composition. Based on the bodyweight of the patent, the dosage may be administered in one or moredoses several times per day or per week. Multiple dosage units may berequired to achieve a therapeutically effective amount. For example, ifthe dosage form is 1000 mg, and the patient weighs 40 kg, one pill willprovide a dose of 25 mg per kg for that patient. It will provide a doseof only 12.5 mg/kg for a 80 kg patient.

[0042] The compounds have shown dose responsiveness in vivo againstviruses and cancers in mice at 500 mg/kg, 2500 mg/kg, 3500 mg/kg, 4000mg/kg, 5000 mg/kg and 6000 mg/kg. Generally a dosage effective in micetranslates to about {fraction (1/12)} of the dosage required in humans.By way of general guidance, for humans a dosage of as little as about 30milligrams (mg) per kilogram (kg) of body weight and up to about 10000mg per kg of body weight is suitable. Preferably from 50 mg/kg to about5000 mg/kg of body weight is used. Most preferably the doses are between100 mg/kg to about 3000 mg/kg of body weight. However, a dosage ofbetween about 2 milligrams (mg) per kilogram (kg) of body weight toabout 400 mg per kg of body weight is also suitable for someindications.

[0043] Intravenously, the most preferred doses may range from about 1 toabout 1000 mg/kg/minute during a constant rate infusion. Benzimidazolederivatives may be administered in a single daily dose, or the totaldaily dosage may be administered in divided doses of two, three, or fourtimes daily. The benzimidazole derivatives are given in one or moredoses on a daily basis or from one to three times a week.

[0044] The benzimidazole derivatives may also be administered inintranasal form via topical use of suitable intranasal vehicles, or viatransdermal routes, using those forms of transdermal skin patches wellknown to those of ordinary skill in that art. To be administered in theform of a transdermal delivery system, the dosage administration will,of course, be continuous rather than intermittant throughout the dosageregimen.

[0045] Generally, the dosage in man is lower than for small warm bloodedmammals such as mice. A dosage unit may comprise a single compound ormixtures thereof with other compounds or other cancer inhibitingcompounds or tumor growth inhibiting compounds or anti-viral compounds.The dosage unit can also comprise diluents, extenders, carriers and thelike. The unit may be in solid or gel form such as pills, tablets,capsules and the like or in liquid form suitable for oral, rectal,topical, intravenous injection or parenteral administration or injectioninto or around the tumor.

[0046] The benzimidazole derivatives are typically mixed with apharmaceutically acceptable carrier. This carrier can be a solid orliquid and the type is generally chosen based on the type ofadministration being used. The active agent can be coadministered in theform of a tablet or capsule, as an agglomerated powder or in a liquidform. Examples of suitable solid carriers include lactose, sucrose,gelatin and agar. Capsule or tablets are easily formulated and can bemade easy to swallow or chew; other solid forms include granules, andbulk powders. Tablets may contain suitable binders, lubricants,diluents, disintegrating agents, coloring agents, flavoring agents,flow-inducing agents, and melting agents. Examples of suitable liquiddosage forms include solutions or suspensions in water, pharmaceuticallyacceptable fats and oils, alcohols or other organic solvents, includingesters, emulsions, syrups or elixirs, suspensions, solutions and/orsuspensions reconstituted from non-effervescent granules andeffervescent preparations reconstituted from effervescent granules. Suchliquid dosage forms may contain, for example, suitable solvents,preservatives, emulsifying agents, suspending agents, diluents,sweeteners, thickeners, and melting agents. Oral dosage forms optionallycontain flavorants and coloring agents. Parenteral and intravenous formswould also include minerals and other materials to make them compatiblewith the type of injection or delivery system chosen.

D. EXAMPLES OF FORMULATION

[0047] The benzimidazole derivatives of this invention are administeredas treatment for cancer and viral infections, including retroviral, byany means that produces contact of the active agent with the agent'ssite of action in the body. The antitumor compounds (active ingredients)of this invention are administered to inhibit tumors by any means thatproduces contact of the active ingredient with the agent's site ofaction in the body of a mammal. They can be administered by anyconventional means available for use in conjunction withpharmaceuticals, either as individual therapeutic agents or in acombination of therapeutic agents. They can be administered alone, butgenerally are administered with a pharmaceutical carrier selected on thebasis of the chosen route of administration and standard pharmaceuticalpractice.

[0048] The benzimidazole derivatives are administered in oral dosageforms as tablets, capsules (each of which includes sustained release ortimed release formulations), pills, powders, granules, elixirs,tinctures, suspensions, syrups, and emulsions. The benzimidazolederivatives may also be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, all using dosageforms well known to those of ordinary skill in the pharmaceutical arts.

[0049] In the methods of the present invention, the compounds hereindescribed in detail can form the active ingredient, and are typicallyadministered in admixture with suitable pharmaceutical diluents,excipients, or carriers (collectively referred to herein as apharmaceutically acceptable carrier or carrier materials) suitablyselected with respect to the intended form of administration, that is,oral tablets, capsules, elixirs, syrups and the like, and consistentwith conventional pharmaceutical practices.

[0050] For instance, for oral administration in the dosage unit form ofa tablet or capsule, the active drug component can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.

[0051] For oral administration in liquid dosage form, the oral drugcomponents are combined with any oral, non-toxic, pharmaceuticallyacceptable inert carrier such as ethanol, glycerol, water, and the like.Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents, and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth, or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes, and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride, and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0052] The benzimidazole derivatives can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamallar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

[0053] Benzimidazole derivatives may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

[0054] The active ingredient can be administered orally in solid dosageforms, such as capsules, tablets, and powders, or in liquid dosageforms, such as elixirs, syrups, and suspensions. It can also beadministered parentally, in sterile liquid dosage forms.

[0055] Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

[0056] Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance. In general, water, asuitable oil, saline, aqueous dextrose (glucose), and related sugarsolutions and glycols such as propylene glycol or polyethylene glycolsare suitable carriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

[0057] Useful pharmaceutical dosage forms for administration of thecompounds of this invention are illustrated as follows:

[0058] Capsules

[0059] A large number of unit capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 to 500 milligrams ofpowdered active ingredient, 5-150 milligrams of lactose, 5-50 milligramsof cellulose, and 6 milligrams magnesium stearate.

[0060] Soft Gelatin Capsules

[0061] A mixture of active ingredient in a digestible oil such assoybean oil, cottonseed oil or olive oil is prepared and injected bymeans of a positive displacement pump into gelatin to form soft gelatincapsules containing 100-500 milligrams of the active ingredient. Thecapsules are washed and dried.

[0062] Tablets

[0063] A large number of tablets are prepared by conventional proceduresso that the dosage unit was 100-500 milligrams of active ingredient, 0.2milligrams of colloidal silicon dioxide, 5 milligrams of magnesiumstearate, 50-275 milligrams of microcrystalline cellulose, 11 milligramsof starch and 98.8 milligrams of lactose. Appropriate coatings may beapplied to increase palatability or delay absorption.

[0064] Injectable

[0065] A parenteral composition suitable for administration by injectionis prepared by stirring 1.5% by weight of active ingredient in 10% byvolume propylene glycol and water. The solution is made isotonic withsodium chloride and sterilized.

[0066] Suspension

[0067] An aqueous suspension is prepared for oral administration so thateach 5 ml contain 100 mg of finely divided active ingredient, 200 mg ofsodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g ofsorbitol solution, U.S.P., and 0.025 ml of vanillin.

[0068] The present invention also includes pharmaceutical kits useful,for example, for the treatment of HIV infection, which comprise one ormore containers containing a pharmaceutical composition comprising atherapeutically effective amount of a benzimidazole derivative. Suchkits may further include, if desired, one or more of variousconventional pharmaceutical kit components, such as, for example,containers with one or more pharmaceutically acceptable carriers,additional containers, etc., as will be readily apparent to thoseskilled in the art. Printed instructions, either as inserts or aslabels, indicating quantities of the components to be administered,guidelines for administration, and/or guidelines for mixing thecomponents, may also be included in the kit. In the present disclosureit should be understood that the specified materials and conditions areimportant in practicing the invention but that unspecified materials andconditions are not excluded so long as they do not prevent the benefitsof the invention from being realized.

[0069] The following examples are illustrative and are not meant to belimiting to the invention.

[0070] Colon, Breast and Lung Tumor Cells Test

[0071] The following cell culture tests were performed to test thetoxicity of the benzimidazole compounds on colon, breast and lung humantumor cells. The viability of the cells were tested by looking at MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)reduction. MTT assay is a well known measure of cell viability.

[0072] The colon tumor cells (HT29 from American Type Culture Collection(ATCC) and the breast cells (MX1 from cell lines from ATCC) werecultured in Eagle's Miminal Essential Medium with 10% fetal bovineserum. The lung tumor cells (A549 from ATCC cell lines) were cultured inHam's F12 medium with 10% fetal bovine serum.

[0073] The tumor cells were passaged and seeded into culture flasks atthe desired cell densities. The culture medium was decanted and the cellsheets were washed twice with phosphate buffered saline (PBS). The cellswere trypsinized and triturated prior to seeding the flasks. Unlessotherwise indicated the cultures were incubated at 37±1° C. in ahumidified atmosphere of 5±1% carbon dioxide in air. The cultures wereincubated until they were 50-80% confluent.

[0074] The cells were subcultured when the flasks were subconfluent. Themedium was aspirated from the flasks and the cell sheets rinsed twicewith PBS. Next, the Trypsin Solution was added to each flask to coverthe cell sheet. The Trypsin Solution was removed after 30-60 seconds andthe flasks were incubated at room temperature for two to six minutes.When 90% of the cells became dislodged, growth medium was added. Thecells were removed by trituration and transferred to a sterilecentrifuge tube. The concentration of cells in the suspension wasdetermined, and an appropriate dilution was made to obtain a density of5000 cells/ml. The cells were subcultured into the designated wells ofthe 96-well bioassay plates (200 microliter cell suspension per well).PBS was added to all the remaining wells to maintain humidity. Theplates were then incubated overnight before test article treatment.

[0075] Each dose of test article was tested by treating quadruplicatewells of cultures with 100 microliter of each dilution. Those wellsdesignated as solvent controls received an additional 100 microliter ofmethanol control; negative controls wells received an additional 100microliters of treatment medium. PBS was added to the remaining wellsnot treated with test article or medium. The plates were then incubatedfor approximately 5 days.

[0076] At the end of the 5 day incubation, each dose group was examinedmicroscopically to assess toxicity. A 0.5 mg/ml dilution of MTT was madein treatment medium, and the dilution was filtered through a 0.45micrometer filter to remove undissolved crystals. The medium wasdecanted from the wells of the bioassy plates. Immediately thereafter,2000 microliter of the filtered MTT solution was added to all test wellsexcept for the two untreated blank test wells. The two blank wellsreceived 200 microliters of treatment medium. The plates were returnedto the incubator for about 3 hours. After incubation, the MTT containingmedium was decanted. Excess medium was added to each well and the plateswere shaken at room temperature for about 2 hours.

[0077] The absorbance at 550 nm (OD₅₅₀) of each well was measured with aMolecular Devices (Menlo Park, Calif.) VMax plate reader.

[0078] The mean OD₅₅₀ of the solvent control wells and that of each testarticle dilution, and that of each of the blank wells and the positivecontrol were calculated. The mean OD₅₅₀ of the blank wells wassubtracted from the mean of the solvent control wells, and test articlewells, respectively to give the corresponding mean OD₅₅₀.${\% \quad {of}\quad {Control}} = {\frac{{corrected}\quad {mean}\quad {OD}_{550}\quad {of}\quad {Test}\quad {Article}\quad {Dilution}}{{corrected}\quad {mean}\quad {of}\quad {OD}_{550}\quad {of}\quad {Solvent}\quad {Control}} \times 100}$

[0079] Dose response curves were prepared as semi-log plots with % ofcontrol on the ordinate (linear) and the test article concentration onthe abscissa (logarithmic). The EC₅₀ was interpolated from the plots foreach test article.

[0080] For the test articles administered in methanol, separateresponses were prepared to correct for the methanol data.3

[0081] Adriamycin was used as a positive control. In all cases, it wasmore toxic than any of the test materials by one or two logs. Adriamycinis one of the more potent agents in current use and one with significantside effects. The peak plasma concentration of other, quite effectivechemotherapeutic agents may be 10 to 50 times higher than that ofAdriamycin.

[0082] The EC₅₀ is the concentration at which one half of the cells arekilled. TABLE 1 EC-50 Result (ppm) Test Material HT29 HT29 MX1 MX1 A549A549 Adriamycin 0.03  0.006 0.02  0.001 0.03  0.009 benomyl 0.742 0.7471.42  2.42  0.980 1.02  carbendazim 0.621 0.662 0.829 0.856 0.856 0.836

[0083] In normal healthy cells, the following results were obtained. Asis evident, the benomyl and carbendazim were much less toxic to normalhealthy cells than adriamycin. TABLE 2 EC-50 Broncheal Kerotinoyle TestMaterial Cells Cells Fibroblasts Benomyl 0.728 0.682  3.26  2.4   3.24 2.81 Carbendazim 0.320 0.506  0.752  0.822  1.52  1.42 Adriamycin 0.0150.0020 0.0035 0.0093 0.065 0.10

[0084] In a related study using lung tumor cells (A-549) breast tumorcells (MCF-7) and colon tumor cells (HT-29), thiabendazole effectivelykilled these cells. Table 3 summarizes the results TABLE 3 OpticalDensity Concentration (ppm) A-549 MCF-7 HT-29 0—Control 0.600 0.2450.398 173   0.007 0.007 0.005 35   0.411 0.025 0.011 17.3 0.851 0.2580.204  3.46 1.12  0.466 0.713  0.87 1.32  0.507 0.852

[0085] These experiments show that these compositions are effective inkilling tumor cells of the breast, colon and lung type.

[0086] Carbendazim has shown broad-scale efficacy against multiplecancer types both in vitro and in vivo. The cancers tested includecolon, lung, breast, prostate, pancreatic, leukemia, melanoma,neuroblastoma, ovarian, neck and head, and brain. Also multiple celllines were tested in almost instances.

[0087] The initial efficacy is comparable to existing best availabledrugs. But with carbendazim, the tumors do not recur or reappear ashappens with Cytoxan and Taxol, which otherwise are quite good againstbreast cancer. Similarly, pancreatic cancer does not appear to come backas often happens with Gemcitabine treatment.

[0088] Carbendazim is particularly good in mouse melanoma in mice, whichmany people believe is the best predictive model for efficacy in humans.It has shown outstanding broad and good results in the human tissuecloning test. This is an in vitro test on conventionally treated andrecently excised human tumors.

[0089] Carbendazim is equally effective against p53 deficient/defectivecell lines, unlike most existing drugs. It appears that carbendaziminduces apoptosis in cancer cells at sub-lethal concentrations to normalcells.

[0090] Other benefits of carbendazim are:

[0091] Its oral LD₅₀ in mice is quite high (11,000 mg/kg), a low overalltoxicity unlike most cancer drugs. For perspective the LD₅₀ of tablesalt is 3750 mg/kg.

[0092] It is effective in cancers that form tumors and those that donot, e.g. both carcinomas and sarcomas.

[0093] The results of these studies are provided in more detail below.

Mechanism of Action Studies

[0094] Some of the pharmacological effects of carbendazim weredemonstrated by studying its ability to induce apoptosis in cancercells, studying its effect on p53-abnormal cell lines and determiningduring which cellular life cycle phase carbendazim exerts its effects.

[0095] Apoptosis Study:

[0096] Apoptosis is a specific type of cell death which differs fromnecrosis and is characterized by specific morphological, biochemical andmicellular cell changes. Abnormalities in p53 expression are generallylinked with the prevention of apoptosis and p53 abnormalities are commonin human tumors which are resistant to conventional cytotoxic agents.

[0097] Summary of test results. The extent of apoptosis is measured inhuman tumor cell lines after treatment for 1, 2, 3 and 4 days withcarbendazim. At each of these time points, the cells are harvested andassayed using the terminal deoxynucleotidyl transferase (TdT) assays.Both microscopy and flow cytometry were used for the TdT assay.

[0098] In MCF7, HT29, B16 and SK-MES cell lines, there was aconcentration dependent effect on cell growth. In most cases atconcentrations of carbendazim greater than 1 μg/ml, the growth rate wassignificantly slower than in the untreated samples. After completion ofthe studies on the MCF7 and HT29 cell lines, it was evident that the 0.1μg/ml concentration had little effect on either apoptosis or cellgrowth. Therefore subsequent assays were with 5 μg/ml instead of 0.1μg/ml.

[0099] The growth of MCF7 (breast cancer) cells was not significantlyaffected by carbendazim below 10 μg/ml as shown by either method.However, at 10 μg/ml the increase in apoptosis was evident at days 3 and4. The increase in apoptosis was low, less than 10% at the highconcentration. After normalization for the cell growth, carbendazim at10 μg/ml had an effect at day 4.

[0100] The growth of HT 29 (colon) cancer cells was not largely sloweddown by carbendazim below 10 μg/ml. Concentration-dependent increase inapoptosis was observed at days 3 and 4, reaching >25% apoptosis in thepresence of 10 μg/ml carbendazim. After normalization for the cellgrowth rates, the concentration-response effect was seen at day 4.

[0101] At all concentrations, carbendazim affected the growth rates inB16 murine melanoma cell line. Some concentration dependent effects onapoptosis were seen at days 1-4 by TdT microscopy and days 2-4 by flowcytometry. The concentration effects at days 1-4 were much more evidentafter normalization of the apoptosis for the growth rates.

[0102] The growth of SK-MES cells was slowed done by all concentrationsof carbendazim past day 1. The percent of apoptosis showed aconcentration-response effect at days 1-4 by microscopic TdT assay andby flow cytometry at days 3 and 4. A normalized graph showed aconcentration response effect of the compound on apoptosis at all days.

[0103] Conclusion: At concentrations less than 10 μg/ml, the greatestresponse to carbendazim was seen in the SK-MES lines (lung), followed byB16 (murine melanoma), HT29 (colon), and MCF7 (breast) cells. The HT29,SK-MES and B16 cell lines express abnormal p53. Accordingly carbendazimcan induce apoptosis in p53 abnormal cell lines.

[0104] Selectivity in Killing p53 Abnormal Cell Lines

[0105] Carbendazim provides in vivo activity against HT29 tumor cellswhich express abnormal p53.

[0106] Summary of test results: Pairs of tumors of the same type werechosen, one expressing normal p53 and the other abnormal p53. Breastlines used were MCF7 for the normal p53, V4B, a MCF7 cells transformedwith an empty vector, and VM4K, an MCF7 cells transfected with a vectorencoding abnormal p53. In the colorectal cancer model HCT116 with anormal p53 was used and DLD-1 was used as the abnormal p53 cell line.The tumor cells were grown the presence and absence of carbendazim for 7days. Cell growth rates were determined in each group by counting thecell numbers daily using a Coulter counter.

[0107] In the breast model at day 7, using 1 μg/ml of carbendazim, cellcounts in all three cell lines were between 50% and 60% of the control.At concentrations of 5 and 10 μg/ml the MCF7 cell counts were 45% and36% of the control. The V4B and VM4K cell lines were less than 10% ofthe respective controls. The data from the VM4K cell line would alsoindicate the carbendazim is again selectively killing the p53 abnormalcells, however, there was also a marked decrease in the cell numbers inthe V4B line carrying only the empty vector. In light of the controversyregarding the p53 status in MCF7 cells, which some researchers claim toexpress abnormal p53, these differences may be more difficult tointerpret.

[0108] At day 7 in the colorectal model, at 1 pig/ml concentrationcarbendazim cell counts in the DLD1 line (abnormal p53) were only 34% ofthe control. The HCT 116 cell line, (normal p53) showed cell counts thatwere 78% of the control suggesting that carbendazim may selectively killp53 abnormal cells. At carbendazim concentrations of 5 and 10 μg/ml,cell counts were less than 10% of the control in both the DLD-1 and HCT116 lines indicating that at the larger concentration, the drug wasequally toxic to cell lines containing abnormal and normal p53.

[0109] Conclusion. At higher concentrations, 5 and 10 μg/ml, the drugwas equally toxic to cell lines containing abnormal p53 and normal p53in both colon and breast cancers.

In vitro Studies of Carbendazim on Cancer Cell Lines

[0110] A dose response effect of carbendazim in a human tumor cloningforming units study (HTCU) is summarized below. This is an in vitro testof treatments on conventionally treated, then recently excised humantumors. It is an important study because carbendazim is showingeffectiveness against cell lines which have survived conventionaltreatment and which themselves have not had undergone too many passages(due to recent excision). This is significant since long living celllines undergo changes (passages), some of which may affect theirresistance to some drugs, and hemotherapeutic agent resistant cells canbe formed. The data show the activity as the tumors tested that had ≦50%survival (a high number is desirable).

[0111] In vitro Human Tumor Colony Forming Units Test

[0112] Solid tumors removed by patients are minced into 2 to 5 mmfragments and immediately placed in McCoy's Medium 5A plus 10% heatinactivated newborn calf serum plus 1% penicillin/streptomycin. Within 4hours, these solid tumors are mechanically disassociated with scissors,forced through No. 100 stainless steel mesh, through 25 gauge needles,and then washed with McCoy's medium as described above. Ascitic,pleural, pericardial fluids and bone marrow are obtained by standardtechniques. The fluid or marrow is placed in sterile containerscontaining 10 units of preservative free heparin per ml. of malignantfluid or marrow. After centrifugation at 150×g for 10 minutes, the cellsare harvested and washed with McCoy's medium plus 10% heat inactivatedcalf serum. The viability of cell suspensions is determined on ahemocytometer with trypan blue.

[0113] Cells to be cloned are suspended in 0.3% agar in enrichedCMRL1066 supplemented with 15% heat inactivated horse serum, penicillin(100 units/ml), streptomycin (2 mg/ml), glutamine (2 mM), insulin (3units/ml), asparagine (0.6 mg/ml), and HEPES buffer (2 mM). For thecontinuous exposure test each compound is added to the above mixture.Cells are placed in 35 mm petri dishes in a top layer of agar over anunderlayer of agar to prevent growth of fibroblasts. Three plates areprepared for each data point. The plates are placed in a 37° C.incubator, and are removed on day 14 for counting of the number ofcolonies in each plate. The number of colonies (defined as 50 cells)formed in the 3 compound treated plates is compared to the number ofcolonies formed in the 3 control plates, and the percent coloniessurviving at the concentration of compound can be estimated. Threepositive control plates are used to determine survival rate. Orthosodiumvanadate at 200 μg/ml is used as the positive control. If there is <30%colonies in the positive control when compared to the untreated control,the test is evaluated. Activity of Carbendazim Against Human TumorColony Forming Units 1 Hour Exposure 1 Hour Exposure Concentration—μg/mlConcentration—μg/ml Tumor Type 0.5 5.0 50 0.5 5.0 10 50 Brain — — — 0/20/2 — 2/2 Breast 0/1 0/1 0/1 0/3 0/4 0/1 2/3 Cervix — — — 0/1 0/1 — 1/1Colon 0/1 0/1 0/1 0/5 1/5 — 2/5 Head & Neck 0/1 0/1 0/1 0/1 0/1 — 1/1Kidney 0/1 0/1 0/1 0/2 0/2 — 1/2 Lung, non-small cell 0/1 0/1 0/1 0/62/6 0/1 2/5 Melanoma 0/2 0/2 1/2 0/3 1/3 — 2/3 Mesothelioma — — — 0/10/1 0/1 — Ovary 0/3 0/3 0/3 0/1  2/13 — 10/13 Sarcoma 0/1 0/1 1/1 0/10/1 — 1/1 Stomach — — — 0/1 0/1 — 1/1 Uterus — — — 0/3 0/3 — 2/3 UnknownPrimary — — — 0/1 0/1 — 0/1  0/11  0/11  2/11 0/4  6/44 0/3 27/41 0% 0%18% 0% 14% 0% 66%

[0114] (4-thiazolyl)-1H-benzimidazole shows efficacy in the Human TumorColony Forming Units test described above when tested using continuousexposure of the cells to the (4-thiazolyl)-1H-benzimidazole.

[0115] The following table summarizes these results showing positiveresults on a number of cancer types: 1 Hour Exposure Continuous ExposureConcentration— Concentration— μg/ml μg/ml Compound 0.5 5.0 50.0 0.5 5.050.0 2-(methoxycarbonylamino) 0/1 0/1 0/1 0/14 3/14 5/14 benzimidazole(4-thiazolyl)-1H- 0/3 0/3 0/3 1/10 2/10 7/10 benzimidazole

In vivo Studies of Carbendazim on Cancer Cell Lines

[0116] The dose response effect of carbendazim in mice infected withvarious cancer types was studied in standard screening tests.

[0117] The results of these studies conducted to study the dosageresponse of carbendazim on various cancer types are summarized below.These results are representative of a number of tests in which a knownchemotherapeutic agent is used as the control so that the efficacy ofthe carbendazim can be compared to it. Efficacy of carbendazim has beenshown in the following cancers:

[0118] In a Prostate cancer model doses of 4000, 5000 and 6000 (mg/kggiven once weekly) were as effective as Mitoxantrone at 70 days. It wasbetter than Cytoxan at these doses of 4000, 5000 and 6000 (mg/kg giventwice weekly) through 40 days. The tables show tumor weight in mg foreach dose. The carbendazim was given once a week (p.o) and theMitoxantrone was given by i.v. (q.d.x5). dose day 1 day 5 day 9 day 12day 16 day 19 peanut oil 61.6  88.9 146.8 184.9 278.1 305.8 control 6000mg/kg 62.1  92.2 140.4 162   226.5  275.99 carbendazim 5000 mg/kg 63.1100.1 116.8 138.3 280.4 246.9 carbendazim 4000 mg/kg 63.1  97.5 159  192.7 282.1 311.6 carbendazim Mitoxantrone 61.9  91.5 120.4 150.9 169.7187.1   1.5 mg/kg

[0119] dose day 23 day 26 day 30 day 33 day 37 day 40 peanut oil 385.6592.4 518   625.2 537.6 594   control 6000 mg/kg 301.9 400.9 416.6 447.3546.3 514.4 carbendazim 5000 mg/kg 281.3 374.6 370.6 428.6 406.4 391.6carbendazim 4000 mg/kg 316.6 368.1 351.3 410.7 506.8 484.9 carbendazimMitoxantrone 208.5 248   247.3 296.9 363   465     1.5 mg/kg

[0120] dose day 44 day 47 day 51 day 53 day 59 day 61 peanut oil 714.1777.4 665.7 764.8 981.3 936   control 6000 mg/kg 505.2 484   438.3 499.8492.2 480.1 carbendazim 5000 mg/kg 445.7 454.7 505.9 543.3 628.6 579.1carbendazim 4000 mg/kg 481.5 511.5 543.1 552.9 507.8 560.3 carbendazimMitoxantrone 545.6 474.9 495.8 566.2 656.8 657.4   1.5 mg/kg

[0121] dose day 65 day 68 day 72 peanut oil control — — — 6000 mg/kgcarbendazim 581.9 525.9 667.3 5000 mg/kg carbendazim 562.3 562.3 602  4000 mg/kg carbendazim 631.2 697.8 739.1 Mitoxantrone 1.5 mg/kg 775.1820.8 707  

[0122] Both the Cytoxan and the carbendazim were given p.o. twice a weekdose day 1 day 5 day 8 day 12 day 15 day 19 peanut oil 66.9 118.2 185.8250.2 264.5 351   control 6000 mg/kg 66.7  97.3 143   193.5 237.9 316.2carbendazim 5000 mg/kg 67.9  84.9 126.8 152.8 184.2 199.5 carbendazim4000 mg/kg 67.2 110.4 157.6 192.6 238.9 298.7 carbendazim Cytoxan 66.7 98.4 179.4 234.6 259.7 278.1  300 mg/kg

[0123] dose day 22 day 26 day 29 day 33 day 36 day 39 peanut oil 416.1446.2 555.3 802.7 868.4 1032.3 control 6000 mg/kg 331.5 371.7 421.7517.2 529.9  595.2 carbendazim 5000 mg/kg 236.8 247.6 293.6 351.5 409.8 497.8 carbendazim 4000 mg/kg 330.5 347.9 346.6 421.1 464.9 517 carbendazim Cytoxan 351.2 467.8 583.8 786.1 904.2 1143.5  300 mg/kg

[0124] In the Colon—HT29 mouse model carbendazim at doses of 4000, 5000and 6000 (mg/kg given twice weekly) it is better than Cytoxan in thismodel. At a dose of 3000 (mg/kg given twice weekly) it is also betterthan Cytoxan.

[0125] The carbendazim and Cytoxan were given twice weekly. dose day 1day 5 day 8 day 12 day 16 day 19 peanut oil 53.5 67.1 98.6 154.6 187  236.3 control Cytoxan 51.3 60.3 42    58.5 64   87.8  300 mg/kg 6000mg/kg 53.5 55.8 45.2  62.8  46.4  59.4 carbendazim 5000 mg/kg 53.5 58  58   104.3  83.2 109.7 carbendazim 4000 mg/kg 51.3 63.8 59.7  85.8  81.1119.4 carbendazim

[0126] dose day 23 day 26 day 29 day 37 day 40 peanut oil control 335.5433.1 499.9 786.3 984.9 Cytoxan 300 mg/kg 137.7 174 252 407.3 503 6000mg/kg carbendazim 77 78.4 60 51.3 62.5 5000 mg/kg carbendazim 129.2149.4 133.3 185.6 185.1 4000 mg/kg carbendazim 142.1 159.6 156.2 184.6212.9

[0127] dose day 1 day 6 day 9 day 13 day 16 day 20 control-no treatment77.1 172.3 231.4 348.1 409.3 478.4 peanut oil control 75.8 172.2 218.2300.3 344 460 Cytoxan 300 mg/kg 76.6 132.5 152.5 142.8 188.1 226 3000mg/kg 75.8 108.1 110.4 141.4 152.3 121.7 carbendazim

[0128] dose day 23 day 26 day 29 control—no treatment 582.8 710.2 867.3peanut oil control 540.8 701.5 863   Cytoxan 300 mg/kg 372.7 375   478  3000 mg/kg carbendazim 141.8 173.5 209.5

[0129] In the Breast—MX-1 model, carbendazim at 4000, 5000, and 6000(mg/kg, twice weekly) was dose responsive in slowing the growth of thetumor and was better than Cytoxan. It is also very effective in MCF-7Lbreast line. At a dose of 3000, (mg/kg given twice weekly) it wasequivalent to Navelbine.

[0130] The Cytoxan and the carbendazim were given twice weekly, p.o.dose day 1 day 5 day 8 day 12 day 15 peanut oil control 70.9 208.6 5261153.6 2267.9 Cytoxan 300 mg/kg 70 32.9 4.2 3.2 0 6000 mg/kg carbendazim70.4 151.8 259.9 492.9 663.6 5000 mg/kg carbendazim 70.1 157.4 272.1535.9 856.4 4000 mg/kg carbendazim 70.3 158 320.4 626.2 1126.5

[0131] When the tumor was shrunk with Taxol first and then thecarbendazim therapy started when the tumor began to grow again on day130, the carbendazim treatment was begun and the tumor subsequentlyshrunk to zero. The Navelbine was given 1.6 mg./kg, qdx5, i.p; the Taxolwas given 16 mg/kg, qdx5, i.p; and the carbendazim was dosed twiceweekly, p.o. The carbendazim treatment was better than Navelbine and asgood as Taxol in this study. dose day 1 day 5 day 8 day 12 day 15 peanutoil control 72.9 91.4 95.2 117.1 121.4 3000 mg/kg carbendazim 70.8 98.790.8 110.1 106.8 Navelbine 68.1 95.3 83.2 106.1 116.3 Taxol 68.9 85.252.1 36.3 27.5

[0132] dose day 20 day 23 day 26 day 29 day 35 day 37 peanut oil 157.6171.9 170.1 211.6 226.1 229.4 control 3000 mg/kg 112.5 137.4 127.9 135.2140.1 137.9 carbendazim Navelbine 135.3 160.5 157.9 162.8 195.8 212.2Taxol 28.5 31.8 32.4 34.2 36.3 32.9

[0133] dose day 41 day 44 day 48 day 51 day 55 day 58 peanut oil 253.2257.7 252.1 247.8 263.9 278.3 control 3000 mg/kg 129.2 128 134.6 111.998.7 107.9 carbendazim Navelbine 223.1 217.4 237.3 222.3 255.8 251.6Taxol 29.9 38.7 29.7 33.3 27.1 35.2

[0134] dose day 62 day 65 day 69 day 72 day 76 day 79 peanut oil 274.4252.8 275.2 277.9 274.7 296.4 control 3000 mg/kg 102.3 103.7 85.1 81.475.2 66.1 carbendazim Navelbine 254.2 257.3 306.1 301.9 307.6 340.7Taxol 35.2 35.2 22.2 33.2 32.9 35.2

[0135] dose day 82 day 85 day 89 day 93 day 97 day 100 peanut oil 277.2276.1 168.7 197.2 278.8 264.6 control 3000 mg/kg 66.3 61.9 18.1 39.353.9 54.2 carbendazim Navelbine 322 352.1 249.9 314.9 375.6 368.6 Taxol35.2 39.2 17 36.8 43.7 46.8

[0136] dose day 103 day 105 day 110 day 113 day 116 day 119 peanut oil266.1 263.1 277.2 278.3 288.9 305.3 control 3000 mg/kg 49.7 52.4 52.449.9 51.3 43.7 carbendazim Navelbine 399.3 391.1 418.8 440.6 544.4 491.9Taxol 53 53.2 50.5 48.4 65.9 65.8

[0137] dose day 124 day 131 day 134 day 137 day 140 day 144 peanut oil331 371.2 396.7 440.5 449.5 482.8 control 3000 mg/kg 43.7 49.2 47.1 53.949.4 52.2 carbendazim Navelbine 514.9 607.3 741.5 692.6 687.4 772.3Taxol 76.6 86.8 92.8 97.9 92.6 92.4

[0138] dose day 147 day 152 day 155 day 158 day 161 day 165 peanut oil506.6 540.7 — control 3000 mg/kg 50.6 53.9 49.4 40.7 49.4 49.4carbendazim Navelbine 811 809.4 — Taxol 104.6 105.9 116 68 73.1 68

[0139] dose day 172 day 175 day 179 day 182 day 186 day 189 peanut oilcontrol 3000 mg/kg 61.2 64.6 69.6 66.3 74.1 81.3 carbendazim NavelbineTaxol 68 71.9 73.1 68.1 67.1 68.1

[0140] dose day 193 day 196 day 200 day 207 day 210 day 214 peanut oilcontrol 3000 mg/kg 78.6 92.2 96.7 117.3 126.2 137 carbendazim NavelbineTaxol 66.3 63.9 58.5 51.4 54.8 34.1

[0141] dose day 217 day 221 day 224 day 228 day 232 peanut oil control3000 mg/kg carbendazim 142.1 162.3 167.3 175.8 209.3 Navelbine Taxol27.5 20.8 20.8 20.8 0

[0142] dose day 236 day 239 day 245 day 249 peanut oil control 3000mg/kg carbendazim 145.7 136.3 158.7 197.1 Navelbine Taxol 0 0 0 0

[0143] In a third study carbendazim was tested in mice MX-1 model withthe mice receiving estrogen tablets. Estrogen accelerates the growth ofthe breast cancer. At doses of 2000, 4000 and 6000 (mg/kg given onceweekly) it was better than both Navelbine (1.6 mg/kg, qdx5, i.p.) andTaxol (16 mg/kg, qdx5, i.p.) in this faster growing MX-1 cancer. doseday 1 day 5 day 9 day 12 day 16 peanut oil control 109.2 307.7 947.61702.8 3359.8 6000 mg/kg carbendazim 109.5 292.7 593.1 1279.6 1261.64000 mg/kg carbendazim 109.5 337.1 664.2 1143.3 1501.5 3000 mg/kgcarbendazim 110.4 312.4 603.6 1068 1502.8 2000 mg/kg carbendazim 110.4342.3 752.3 1447.6 1609.1 Navelbine 110.2 278.6 874.5 1528.5 2746.8Taxol 110.4 292.7 484.9 876.6 1941.5

[0144] In the Pancreas (Mia-PaCa) model, carbendazim at 3000 and 4000(mg/kg, twice weekly) is as good as or better than gemcitabine. At 2000mg/kg the carbendazim was not as effective after 21 days. Gemcitabinewas given on days 1,4,7 and 10 i.p.; the carbendazim was given p.o.twice weekly. dose day 1 day 5 day 9 day 12 day 16 peanut oil control63.1 118.5 186.6 228.4 294.6 4000 mg/kg carbendazim 64 78.9 121.3 113.4133.4 3000 mg/kg carbendazim 63.1 71.8 100.1 100.4 139.6 2000 mg/kgcarbendazim 63.7 85.2 128.4 155.1 213.4 Gemcitibine −80 mg/kg 63.9 71.781.7 77.1 94.9

[0145] dose day 19 day 23 day 26 day 30 day 33 day 37 peanut oil 325.9462.8 489.5 546.6 control 4000 mg/kg 119.4 157 154.2 124.3 131.4 129.2carbendazim 3000 mg/kg 131.9 146.6 131.9 140.1 135.1 110.6 carbendazim2000 mg/kg 182.2 185 189.9 214.1 206.7 217.6 carbendazim Gemcitibine111.3 167.1 204 258.7 330.3 404.3 −80 mg/kg

[0146] dose day 40 day 44 day 47 day 51 day 54 day 61 peanut oil control4000 mg/kg carbendazim 3000 mg/kg 105 105 120.1 124.1 124.1 118.6carbendazim 2000 mg/kg 214.55 214.5 210.9 222.4 225.6 214.1 carbendazimGemcitibine 503.7 −80 mg/kg

[0147] dose day 65 day 68 day 72 day 75 day 79 peanut oil control 4000mg/kg carbendazim 3000 mg/kg carbendazim 130 130 69.2 52.5 75.8 2000mg/kg carbendazim 217.3 217.3 202.6 178.2 169.7 Gemcitibine −80 mg/kg

[0148] In the Panc-01 model for pancreatic cancer carbendazim at a doseof 5000 mg/kg was better than Gemcitabine at 32 days. The Gemcitabine isgiven i.p., q3dx4 and the carbendazim is given p.o. twice weekly to theend. dose day 1 day 4 day 8 day 11 day 18 day 22 control-no treatment64.1 110.8 201.5 339.7 726 1001.4 peanut oil control 64.3 123.8 200.3306 740.3 1174.1 Gemcitabine 80 mg/kg 64 106.3 171.7 248.5 561.4 943.65000 mg/kg 64.4 115.5 166.8 247 417.9 574.3 carbendazim

[0149] dose day 25 day 29 day 32 control—no treatment 1183   peanut oilcontrol 1126.7 Gemcitabine 80 mg/kg 1053   1183 5000 mg/kg carbendazim 695.8  845 807.9

[0150] In the Neuroblastoma (SK-N-MC) model a decrease in tumor growthat 5000(mg/kg) is shown and there is favorable activity early in thestudy compared to topotecan. It is expected, based on learning from theother studies, that a lower dose would be effective. The topotecan isgiven i.p. qdx5 and the carbendazim is given p/.o. twice weekly. doseday 1 day 5 day 9 day 12 day 16 day 19 peanut oil 454.8 1034.1 1533.11141.8 1402 1668.2 control 5000 mg/kg 474.4 505.8 799.3 841.8 732 1424.3carbendazim Topotecan 3 504.9 209.8 57.5 71.8 144.7 294.6 mg/kg

[0151] dose day 23 day 25 day 31 peanut oil control 1852.8 2025   5000mg/kg carbendazim 1480.5 936  Topotecan 3 mg/kg  648.1  886.6 1150.2

[0152] In the Rhabdomysarcoma model, a childhood cancer, at day 29carbendazim at doses of 3000, 4000 and 5000 (mg/kg given once weekly,p.o.) is quite effective against this tumor with no toxicity in the 3000(mg/kg) group. Topotecan (i.p., qdx5) is quite active against thismodel. dose day 1 day 5 day 8 day 12 day 15 peanut oil control 58.1121.3 177 223 340.9 5000 mg/kg carbendazim 58.1 95.1 87 88.1 106.4 4000mg/kg carbendazim 59.5 110.7 124.2 121.6 155.3 3000 mg/kg carbendazim58.2 110.4 136.6 176.7 248.2 Topotecan 3 mg/kg 58.3 82 53.3 24.1 26.6

[0153] dose day 19 day 22 day 26 day 29 day 35 day 37 peanut oil 558689.3 894 948.3 1038.2 1098.5 control 4000 mg/kg 112.6 107.9 137.5 174.3238.8 317.4 carbendazim 3000 mg/kg 113 127 140.8 144.3 131.9 152.4carbendazim 2000 mg/kg 313.1 291.2 384.3 417.4 591.2 492.9 carbendazimTopotecan 13.4 18.9 40.9 64.2 107.9 158.9 3 mg/kg

[0154] dose day 40 day 43 day 47 peanut oil control 4000 mg/kgcarbendazim 313.9 423   171.5 3000 mg/kg carbendazim 142.6 195.5 235.82000 mg/kg carbendazim 555.6 682.1 854.3 Topotecan 3 mg/kg 184.4 279.2351.9

[0155] In the Lung cancer model MV522 all groups received Taxol and thenreceived treatments shown starting 10 days later. At doses of 2000,3000, 4000 and 5000 (mg/kg given twice weekly, p.o.) tumor growth wassuppressed in a dose response and the tumor was shrunk in the 5000 mg/kggroup. Day 1 is the start of the treatment with carbendazim. dose day 1day 4 day 8 day 11 day 15 day 18 control - 6.6 5.6 7.3 18.3 39.6 80.2 notreatment peanut oil 6.7 16.9 29.7 50.6 84.9 140.6 control 5000 mg/kg6.5 7.1 16.1 12.7 18.8 27.2 carbendazim 4000 mg/kg 6.6 8.5 17.6 16.220.7 26.6 carbendazim 3000 mg/kg 6.5 7.1 11.9 9.2 14.1 16 carbendazim2000 mg/kg 6.5 10.3 19.9 33.8 44.8 40.5 carbendazim

[0156] dose day 22 day 25 day 29 day 32 day 36 control - 128.8 180.1270.7 307.3 594.6 no treatment peanut oil 233.9 341.4 488.7 control 5000mg/kg 21.5 31.4 40.5 29.5 54 carbendazim 4000 mg/kg 40.8 39.7 58.9 47.362.5 carbendazim 3000 mg/kg 19.9 23.4 37.5 28.9 44.5 carbendazim 2000mg/kg 37.9 43.5 49.8 41.1 53 carbendazim

[0157] dose day 39 day 42 day 46 day 49 day 52 day 56 control - 572.4670.1 no treatment peanut oil control 5000 mg/kg 63 86.8 86 1 0.5carbendazim 4000 mg/kg 62.5 32 carbendazim 3000 mg/kg 59.2 69.3 72.6160.7 207.1 267.4 carbendazim 2000 mg/kg 42.9 47.1 60.3 107.4 107.4175.3 carbendazim

[0158] dose day 59 day 63 day 66 control—no treatment peanut oil control5000 mg/kg carbendazim 4000 mg/kg carbendazim 3000 mg/kg carbendazim 75.4 77   94.5 2000 mg/kg carbendazim 214.9 193.7 188.9

[0159] In a Medulloblastoma model (IMR32) a dosage dependent effect isseen with carbendazim at doses of 5000, 4000, and 3000 (mg/kg, p.o.twice weekly). It is compared with Topotecan (i.p. qdx5). dose day 1 day7 day 9 day 12 day 15 day 19 peanut oil 57.6 82.1 103.8 107.5 143.1182.7 control 5000 mg/kg 57.2 74.9 86.4 94.6 102 125.4 carbendazim 4000mg/kg 58.1 89.3 108 112.9 152.9 162.3 carbendazim 3000 mg/kg 58.1 73.899.3 105.1 119.7 153.4 carbendazim Topotecan 57.8 22.6 23.6 15.7 3.6 3mg/kg

[0160] In the Murine Melanoma—B16 mice model doses of 4000, 5000 and6000 (mg/kg) were at least equal in tumor suppression compared tocytoxan at 30-60 days. Since this tumor is a liquid tumor, there is nochange in tumor weight. The results are summarized in Table 1 below.

[0161] In the P-388 model for leukemia there was a dose responsiveeffect and the results were good. See Tables 2 and 3.

[0162] The data in Tables 1, 2 and 3 are reported in T/C which isinterpreted using the following scale: T/C < 125 no activity T/C =125-150 weak activity T/C = 150-200 modest activity T/C = 200-300 highactivity T/C = 300 with long term survivors excellent activity

[0163] long term survivors for P388 is >30 days, for B16 it is >60 days.The NCI Measure of Success is T/C=125.

Use of Carbendazim in the Treatment of B16

[0164] This study was performed in black mice injected i.p. with B-16mouse melanoma which many researchers believe is the most predictivemodel for efficacy in humans. Carbendazim was equally effective asCytoxan in this model at treating B-16. The activity is dose responsive.Death is the end point in this model. TABLE 1 Dosage (mg/kg) twiceweekly T/C weight change (%) Positive Control 5000 198 −0.64 Cytoxan at300 mg/kg 2500 169 +3.16 one dose 2000 169 −11.63  T/C 191, −8.43% 1250124 +1.99 weight change 1000 176 −4.75  500 141 −0.64

[0165] TABLE 2 In a repeat test the following results were achieved:Dosage (mg/kg) twice weight weekly T/C change (%) Positive Control 6000183 −0.38 Cytoxan at 300 mg/kg 5000 167 −1.44 one dose 4000 138 +3.86T/C 161, −4.28% weight change

[0166] TABLE 3 Use of Carbendazim in the treatment of P388 Dosage(mg/kg) T/C weight change (%) Positive Control 4000 189 −9 cytoxan at125 mg/kg 2000 148 +8 is curative; weight 1000 129 +15 change −14

[0167] Accordingly, carbendazim retards the growth of both solid andliquid tumors in vivo.

[0168] These studies confirm previous in vivo studies in whichcarbendazim was tested in various in vivo models for different types ofcancer.

[0169] Additional in vivo cancer studies are presented in a tabular formbelow. The data is presented in the following format—dose regimen inparenthesis, dosage in mg/kg: tumor growth suppression (tg supp.), toxicdeaths/number of test subjects (deaths), % weight change (wt). TABLE 4Cancer Positive Control dose tg supp. deaths wt. dose tg supp. deathswt. MX1 xenograft- (twice weekly) Cytoxan (1 dose) 6000 70% 1/9  −4 300complete 0/10 −7 5000 63% 1/10 −4 shrinkage 4000 48% 0/10 −1 NCI Measureof Success: 58% MCF-7L (breast) (twice weekly) Cytoxan (1 dose) 600010/10 300 100% 3/10 −15 5000 100%  7/10 −8 NCI Measure of Success: 58%4000 94%  5/10 −7 DU-145 (twice weekly) Cytoxan (1 dose) 6000 33% 1/10−6 300 0% 0/10 +6 5000  48%, 1/10 −8 NCI Measure of Success: 58% 400052% 0/10 −5 A549 (lung) Cytoxan 2500 10/10 125 69% 0/10 −1 500 57%  0/10+10 HT29 (colon) (twice weekly) Cytoxan (1 dose) 6000 97% 9/10 −25 30045% 0/10 +10 5000 79% 2/10 −8 NCI Measure of Success: 58% 4000 78% 3/10−12 3000 65% 2/10 −6 2000 36% 0/10 −3 SK-MES (twice weekly) Taxol (5doses) 6000 69% 3/10 −2 20 75% 0/10 +1 5000 44% 1/10 +2 4000 45% 2/10 +3Navelbine (5 doses) 2 26% 1/10 −5 NCI Measure of Success: 58%

[0170] Carbendazim demonstrated the ability to reduce tumor growth inmice models for lung, colon, murine melanoma and leukemia. The data aresummarized in Table 5 and Table 6 below. TABLE 5 Tumor Positive GrowthControl Tumor Decrease (Dosage in Growth Cancer Dosage (%) mg/kg)Decrease MXI-breast 500 mg/kg 42% cytoxan (125) regression 2500 mg/kg37% A549-lung 500 mg/kg* 57% cytoxan (125) 69% HT29-colon 2500 mg/kg**54% cis-plat (4) 59%

[0171] In the same test 2-(4-thiazolyl)-1H-benzimidazole showed noactivity against MXI breast cancer tumors implanted subcutaneously underthe mice skin. TABLE 6 Increased Positive Cancer Dosage Life SpanControl P388-luekemia 1000 mg/kg 129% cytoxan (125) curative 2000 mg/kg148% 4000 mg/kg decreased life span  −9% B16 melanoma 1000 mg/kg 131%navel- increased bine (2) life span -265% 2000 mg/kg 163% 4000 mg/kg187%

[0172] In the same test 2-(4-thiazolyl)-1H-benzimidazole showed noactivity against P388.

[0173] In the same test 2-(4-thiazolyl)-1H-benzimidazole showed noactivity against B16.

[0174] The in vivo and in vitro data support the assertion thatcarbendazim has broad scale efficacy against multiple cancer types.

[0175] Initial efficacy of carbendazim appears to be comparable to thebest available drugs used for the treatment of any particular cancertype. Furthermore, with continuous treatment, breast cancers do not comeback as usually happens with cytoxan and taxol in breast cancer orgemcitabine in pancreatic cancer.

[0176] It is equally effective against p53 deficient/defective celllines, unlike most existing cancer treatment drugs.

[0177] It is excellent in B16 mouse melanoma, which is believed by manypeople to be the best predictive model for efficacy in humans.

[0178] These same benzimidazole derivatives are effective againstviruses including HIV, influenza, rhinoviruses and herpes viruses. Thebenzimidazole derivatives can be used alone or in combination with otherfungicides.

[0179] The following examples illustrate the effectiveness ofthiabendazole, 2-(4-thiazolyl)-1H-benzimidazole, against HIV and thebenzimidazoles derivatives against a number of viruses.

[0180] The results of these HIV studies are summarized in more detailbelow:

[0181] Thiabendazole is effective at totally suppressing virusproduction in chronically infected cells. The extra cellular viral countgoes effectively to zero or non-detectable levels. Thiabendazole doesnot kill the chronically infected cells though it does reduce the rateof cell proliferation at active concentrations. Thiabendazole does notaffect CD₄ expression in uninfected cells. At effective concentrationsthiabendazole slows but does not alter the normal cellular RNA orprotein synthesis of either infected or non-infected cells.Thiabendazole is effective in a variety of chronically infected celltypes (this effect is not cell type specific.)

[0182] Thiabendazole is effective against a variety of HIV virusstrains. (Not virus strain specific—although some variance by strain isobserved; SK-1>III>B>RF) Also thiabendazole is not effective on SIV invitro or in vivo.

[0183] After 20 months no resistant virus strains to thiabendazole havedeveloped in tests designed to do so. Resistance develops in six monthsor less in this test for existing HIV drugs with resistance strains forprotease inhibitors developing in about 3-4 months.

[0184] Thiabendazole does not adversely affect the activity of existingHIV drugs, AZT, 3TC, ddC, ddI or protease inhibitors (saquinavir andindinavir) in acutely infected cells, nor do any of these existing drugsinterfere with the efficacy of thiabendazole in chronically infectedcells. It is used in combination with these drugs. Thiabendazole is alsoeffective against protease inhibitor resistant viruses.

[0185] Thiabendazole confers temporary suppression of viral productionfrom 4 to 80 days after treatment stops. This is unique and a usefulfeature whenever one has problems with compliance.

[0186] The results of these studies are summarized in detail below:

HIV Virus Replication Study

[0187] Thiabendazole was tested in chronically infected HIV virus. Thesecell populations contain integrated copies of the HIV genome andconstituitively produce HIV at relatively high levels (CEM-SK1, U937-SK1and H9-SK1 from Frederick Research Center, Maryland) or are latentlyinfected and only produce virus after stimulation with phorbol esters,tumor necrosis factor or IL6 (U1 and ACH2). Virus production was reducedin all cell lines tested and thiabendazole did not stimulate virusproduction from the latently infected cells. Reductions in virusproduction were observed when quantifying supernatant reversetranscriptase activity, supernatant p24 as well as intracellular p24,indicating the compound inhibits virus production at a step ofreplication prior to production of intracellular proteins.

[0188] Quantification of the infectivity of virions produced from theinfected cells demonstrates reductions in the number of infectiousvirions in parallel with reductions in supernatant RT or p24, indicatingthe compound reduces the amount of virus produced, but not the qualityof the virions. Inhibition of virus production from the chronicallyinfected cells was observed at concentrations which were nontoxic to thetarget class. Thiabendazole inhibited virus production at concentrationsgreater than 1-10 μg/ml.

[0189] Toxicity to the chronically infected cells was similar to thatobserved with the uninfected cells. Evaluation of thiabendazole onchronically infected cells was performed by evaluation of thymidine(DNA), uridine (RNA) and leucine (protein) incorporation into cellularmacromolecules. Inhibition of cellular macromolecule synthesisparalleled the toxicity of the compound as would be expected and did notoccur at lower nontoxic concentrations found to inhibit virus productionfrom the chronically infected cells.

[0190] After 28 days of treatment on chronically infected cells, thetoxicity of the compound to the target cells appeared similar in bothuninfected and chronically infected cells. The compound does notpreferentially kill HIV-infected cells. Reductions in the level of virusproduction were stable and were observed at concentration greater than10 μg/ml for thiabendazole.

[0191] These results suggest thiabendazole can quickly reduce the levelof virus production from cell populations chronically infected withHIV-1 and the antiviral effect is maintained with prolonged compoundexposure. This reduction of virus production occurs at concentrationswhich are nontoxic to the host cell and which have no effect on thesynthesis of cellular DNA, RNA and protein.

Virus Resistance Studies

[0192] Chronically infected HIV cells were cultured in the presence ofthiabendazole at 1 μg/ml for the first month, 5 μg/ml for the secondmonth, 10 μg/ml for the third month, 20 and 40 μg/ml for the fourthmonth and 80 μg/ml for the fifth and sixth months. At the end of eachmonth, the cells were evaluated for virus production compared tochronically infected cells not treated with the compound. For each ofthe six months of treatment experience, no change in the antiviraleffect of the compound was noticed and the toxicity of the compoundremains identical. Thiabendazole remains active against HIV and thatresistance was not rapidly achieved via the selection of resistantviruses or adaptation of the cells to prevent compound induced toxicity.Virus production remains totally suppressed from cultures treated withthiabendazole at 40 and 80 μg/ml.

[0193] The present invention includes a method of treatment HIV with thebenzimidazole compounds without inducing formation of benzimidazolederivative or thiabendazole resistant HIV.

Reappearance of Virus Production from Chronically Infected CellsPreviously Treated

[0194] Chronically infected cells which were treated with compound forprolonged periods were washed free of compound and cultured to determineif, and when, virus production would resume. Cultures in which treatmentresulted in the total elimination of virus production were used in theseassays. These cultures included chronically infected cells cultured inthe presence of 20, 40, and 80 μg/ml of thiabendazole. Within 4 daysvirus production resumed from the cells cultured in the presence of thelower concentrations of thiabendazole (20 μg/ml and 4 μg/ml). Virusproduction resumed at the 40 μg/ml concentration of thiabendazole by day12. At the highest concentrations virus production was observed atapproximately day 70.

[0195] The present invention includes a method of treatment HIV with thebenzimidazole derivative or thiabendazole and delaying the reappearanceof HIV in plasma following initial treatment of HIV with an antiviralagent or thiabendazole.

Infectability of Cells Treated with Thiabendazole

[0196] Cells which were pretreated with thiabendazole for a long periodof time were washed free of compound and used as a target cellpopulation. The cells were split into 3 populations and labeled Group 1,2 or 3. Group 1 was treated with the compound for 24 hours (at the sameconcentration used in the prolonged treatment phase), washed free ofcompound and cultured in the presence of infectious virus and freshcompound. Group 2 was pretreated for 24 hours, washed free of compoundand cultured in the presence of infectious virus only. Group 3 wascultured for both the pretreatment and the infection phases in freshmedium only (no virus or compound). Virus production from the cellpopulations was identical irrespective of the culture conditions. Theseresults indicate that the chronically infected cells treated forprolonged periods were not super-infected with HIV.

Additional Chronic HIV studies

[0197] Chronic HIV-1 infected cells U1 were derived from an acute HIV-1infection of the promonocytic cell line, U937. The chronic HIV-1infected cells, ACH-2 were derived from an acute HIV-1 infection of theT cell line, A3.01.

[0198] These cells were cultured in medium and the phorbol ester, PMA.PMA causes the cells (both U1 and ACH-2) to be activated and not dividebut it also causes the U-1 cells to differentiate. This results in fewercells in the PMA-treated cultures than the media alone cultures. Cellviability was measured when these cell lines were treated with the testcompound.

[0199] Both cell lines constituitively produce a small amount of HIV-1.ACH-2 cell lines tend to produce more HIV-1 than U1 cells as shown byp-24 ELISA. When either cell line is cultured in the presence of PMAthere is an increase in the quantity of HIV-1 produced as measured bythe p-24 antigen ELISA.

[0200] In addition, the number of institute positive HIV mRNA expressingcells per microscopic field is measured. Comparisons can be made fromthese numbers since the same number of cells were adhered to the glassslides for each drug concentration (10×10⁶ cells/ml).

[0201] These cells were treated with test samples. Thiabendazole at 60μg/ml suppressed replication in the HIV monocytes by 74% and the T-cellHIV replication was increased by 26%. The positive control wasinterferon which suppressed HIV monocytes replication by 80%. AZT showedno activity in this model.

[0202] 2-(Methoxycarbonylamino)benzimidazole suppressed replication inthe HIV monocytes by 9% and the T-cell HIV replication was increased by44%. The positive control was interferon which suppressed HIV monocytesreplication by 80% and suppressed T-cell HIV replication by 60%.

Acute HIV Testing

[0203] In an in vitro acute model for HUV2-(methoxycarbonylamino)benzimidazole inhibited viral replication by100% at 4 μg/ml and AZT inhibited viral replication by 98% at 1 μg/ml.2-(4-thiazolyl)-1H-benzimidazole inhibited viral replication by 98% at60 μg/ml.

[0204] The therapeutic index (TI), the ratio of the toxic dose of drugto efficacious dose of drug for 2-(4-thiazolyl)-1H-benzimidazole is 2.8versus 12, 500 for AZT. The TI for 2-(methoxycarbonylamino)benzimidazoleis 1.8.

In vivo Herpes

[0205] In an in vivo herpes screening test of2-(4-thiazolyl)-1H-benzimidazole at a dose of 200 mg/kg dose, 10% of themice survived with a 10.4 mean death date; at 100 mg/kg dose 50% of themice survived with a 9.2 mean death date. The positive control wasacyclovir at 75 mg/kg dose; 60% of the mice survived with a mean deathdate of 17.2 days. In the same test2-(methoxycarbonylamino)benzimidazole showed no activity.

Other Tests

[0206] Both 2-(4-thiazolyl)-1H-benzimidazole and2-(methoxycarbonylamino) benzimidazole were tested in an in vitroinfluenza model and showed no activity.

[0207] In an in vivo model for influenza2-(4-thiazolyl)-1H-benzimidazole was tested at 200 mg/kg, 67% of themice survived with a mean death date of 8 days; at 100 mg/kg, 62%survived with a mean death date of 8.7 days. The positive control wasamantadine (75 mg/kg) with 100% of the mice surviving for 21 days.2-(Methoxycarbonylamino) benzimidazole was not active in the same test.

[0208] Both 2-(4-thiazolyl)-1H-benzimidazole and2-(methoxycarbonylamino) benzimidazole were tested in an in vitro herpesmodel and showed no activity.

[0209] Both 2-(4-thiazolyl)-1H-benzimidazole and2-(methoxycarbonylamino) benzimidazole were tested in an in vitrorhinovirus model and compared to A-36683. The therapeutic index (TI),the ratio of the toxic dose of drug to efficacious dose of drug, for2-(4-thiazolyl)-1H-benzimidazole is 1-2 and for 2-(methoxycarbonylamino)benzimidazole is 1-3 versus 1000-3200 for A-36683.

[0210] The demonstrated effectiveness of the compounds of the presentinvention in the human breast and lung tumor xenograft models indicatethat the compounds of the present invention are useful for the treatmentof solid tumors in man, and, in particular, tumors of the breast andlung. This conclusion is further supported by published analysescorrelating pre-clinical test results with clinical efficacy ofanti-cancer agents. For example, see: Goldin and Venditti (1980) RecentResults Cancer Research 76: 176-191; Goldin et al. (1981) Eur. J. Cancer17: 129-142; Mattern et al. (1988) Cancer and Metastasis Review 7:263-284; Jackson et al. (1990) Cancer Investigations 8: 39-47. Based onthese published analyses, the exceptional high level of antitumoractivity exhibited by the presently claimed compounds provide strongevidence that the compounds claimed in present invention havetherapeutic utility in the treatment of cancer in man and that they willimprove the quality of life of the patient.

What is claimed is:
 1. A method for treating breast cancer in a patientin need thereof, the method comprising administering to a patient atherapeutically effective amount of a composition comprising abenzimidazole compound of the formula:

wherein, X is hydrogen, halogen, alkyl of less than 7 carbon atoms, oralkoxy of less than 7 carbon atoms; n is a positive integer of less than4; Y is hydrogen, chloro, nitro, methyl, ethyl, or oxychloro; R ishydrogen, an alkyl group of from 1 to 8 carbon atoms, oralkylaminocarbonyl wherein the alkyl group has from 3 to 6 carbon atoms;and R₂ is NHCOOR₁, wherein R₁ is an aliphatic hydrocarbon of less than 7carbon atoms; or a pharmaceutically acceptable salt thereof, or mixturesthereof, wherein the breast cancer had survived treatment with anotheranticancer agent prior to treatment with the benzimidazole.
 2. Themethod of claim 1 wherein said benzimidazole compound has the formula:

wherein R is hydrogen; and R₂ is NHCOOR₁ wherein R₁ is methyl, ethyl orisopropyl.
 3. The method of claim 1 wherein said pharmaceuticallyacceptable salt is a hydrochloride salt.
 4. The method of claim 1wherein said benzimidazole compound is in the form of a parenteralcomposition suitable for administration by injection.
 5. A method fortreating breast cancer in a patient in need thereof, the methodcomprising administering to the patient a therapeutically effectiveamount of a composition comprising 2-(methoxycarbonylamino)benzimidazolewherein the breast cancer had survived treatment with another anticanceragent prior to treatment with the benzimidazole.
 6. The method of claim5 wherein said 2-(methoxycarbonylamino)benzimidazole is in the form of apharmaceutically acceptable salt thereof.
 7. The method of claim 6wherein said pharmaceutically acceptable salt is a hydrochloride salt.